Mitochondria, Oxidative Stress, and the Pathophysiology of Type 2 Diabetes

1. Mitochondria, Oxidative Stress, and the Pathophysiology of Type 2 Diabetes

2. Total Prevalence of Diabetesin the United States(All Ages, 2005):Total: 20.8 million people(7 percent of the population)Diagnosed: 14.6 million peopleUndiagnosed: 6.2 million peopleIncidenceof Diabetes, United States:In 2005: 1.5 million new cases of diabetes were diagnosed in people aged 20 years or olderCost of diabetes in the United States:Total (direct and indirect : $132 billionDirect medical costs: $92 billionIndirect costs: $40 billion (disability, work loss, premature mortality). The Ascent of Man ( and Type 2 Diabetes ) Sloth or Gluttony ? (from the National Diabetes Information Clearing House)

3. Human Migration According to Mitochondrial DNA Diaspora(Pure Motherly Lineages) Mitochondrial Eve ? South America Western Europe Africa (upside down) North America You are here  India Carb-free China Russia Food Quest Australia MAP of human race migrations, according to the Mitochondrial DNA. The Numbers represent thousands of years before present time (time units used in Radiocarbon dating). The Blue line represents area covered in ice or tundra during the last great ice age. The Letters represent mitochondrial DNA haplotypes (pure maternal lineages); Haplogroups can be used to define genetic populations and are often geographically oriented. Fuel gathering and energy expenditure

4. What ARE Mitochondria ? EndoSymbiotic Bacteria “The Little Friends Within” • Maternally Inherited Co-Incident DNA • Aerobic Cellular Respiration (90%) • Chloroplast’s Gas Exchanger (O2)(CO2) • Signaling System The “ PowerHouses of the Cell ” • Oxidative - Phosphorylative Coupling • Endothermic Heat Source • Generation of ATP

5. Mitochondrial ChemiosmosisThe BioEnergetics of ATP Generation H+ H+ H+ ATP ADP +iP H+ H+ H+ Cytosol Cytosol H+

6. Oxidative - Phosphorylation H+ H+ H+ H+  H+ H+ H+ H+ ATP COUPLING: ATP synthesis (phosphorylation) must be coupled to the generation of a H+ reservoir/pH gradient (oxidation) between the mitochondrial membranes.

7. Elemental H+  ATP ADP + iP (phosphate) Remember this ATP-Synthase

8. ATP-Synthase:Hydrogen-Powered H+ H+ H+ H+

9. What Causes Insulin Resistance? What Causes Pancreatic ßetaCell Impairment?

10. Insulin Resistance:Skeletal Muscle • Triglyceride Content of skeletal muscle is directly proportional to Insulin Resistance • Increased Circulating Free Fatty Acids (FFAs) result in suppression • of Insulin-mediated Glucose Uptake • Triglyceride accumulates either by • 1) Fat overload or • 2) Diminished ß-oxidation Intramyocellular Lipid (IMCL) more closely correlates with insulin resistance than BMI, waist-to-hip ratios, or total body fat(mcGarry)

11. Which Came First?Insulin Resistance or Fatty Acid accumulation? • With hyperinsulinemic-euglycemic clamp, • Artificial elevation of plasma FreeFatty Acid • concentration in otherwise healthy humans leads to: • 22-61% increase in Intramyocellular Lipid content (Brechtel et al. 2001) • 40-50% loss of either oxidative glucose disposal or Muscle glycogen synthesis (Boden 1995) • With hyperinsulinemic-euglycemic clamp, • Glucose infusions in healthy, young, lean yet insulin resistant • Offspring of patients with type-2 diabetes: • 60% decrease in glucose uptake within skeletal muscle insulin-resistant offspring of T2DM parents • 80% increase in the intramyocellular lipid content (IMCL) • Co-incident 30% reduction in mitochondrial phosphorylation (Petersen et.al. NEJM 2004)

12. Mitochondrial Dysfunction Loss of Oxidative-Phosphorylation with subsequent declines in ATP generation Petersen et.al. PLoS 2005 MRS (magnetic resonance spectroscopy) assessment of ATP-Synthase flux and intramyocellular inorganic phospate in healthy,normoglycemic (i.e. not “diabetic”) lean Insulin-resistant offspring of T2DM patients versus non-insulin resistant controls Results: Insulin stimulated Glucose uptake declined 50% in IR group 2-Fold increase in IntraMyocellularLipid Content in IR Group 90% increrase in ATP synthesis rate in Control Group vs. 5% increase in mitochondrial ATP synthesis rate in IR Group Insulin-induced phosphate increases correlated with insulin-stimulated increases in ATP synthesis in both groups

13. Mechanism of InAction Loss of Mitochondrial phosphorylation results in the accumulation of triglycerides and other Fatty Acid metabolites, particularly DAGs, Fatty AcylCoA, and ceramides that embed in the cellular membrane leading to a co-incident loss of insulin sensitivity. Deep pathologic mechanism involves the activation of Protein Kinaase C-Theta (PKC-) and the serine/threonine phosphorylation of the Insulin-Receptor Substrate (IRS-1) inhibitting GLUT4 translocation resulting in decreased glucose removal from bloodstream Morino et.al. 2006

14. Pancreatic ßetaCell:Glucose sensing, Mitochondria, and Insulin release [1] [2] Beta-cell mitochondria serve as fuel sensors that link glucose exposure to insulin release: • GLUT2 transporters introduce glucose to mitochondria causing increased ATP:ADP ratio • K+ gate closure--> depolarization • Voltage sensitive Ca++ channels open • Exocytosis of insulin storage granules to bloodstream GLUT2 K+ ATP: ADP Ca++ [4] Insulin [3]

15. ß-Cell Impairment:Loss of glucose-stimulated insulin release Superoxide radicals induce UCP-2 upregulation contributing to proton leak, ATP:ADP, and loss of glucose stimulated insulin release: Hyperglycemia/Fatty acid excess causes chemi-osmotic overload and superoxide (O2-) generation O2- as oxidative SIGNAL, upregulates UCP-2 with decreased ATP production Resulting in loss of insulin secretory response UCP-2 upregulation decreases SO- generartion = tight negative feedback ATP : ADP (SuperOxide Radical)

16. WAKE UP !!!

17. Uncoupling Protein 1 (UCP1) • Produced in brown adipose tissue of newborn humans and hibernating mammals. (we eventually lose our “baby fat” ) Unique to mammals. • Diverts energy from ATP generation to Adaptive Thermogenesis (Heat Release) Proton partioning • Thermogenic uncoupling occurs in response to overeating and cold • Decreases mitochondrial production of Oxidative damage • Adaptive response to cold temperature mediated by norepinephrine with secondary effect of regulating energy balance. • Increased expression due to Fatty acid-induced Superoxide generation from mitochondria. Inhibited by purine nucletide. • NOT found in skeletal muscle. • UCP1 expression in the smooth muscle cells can increase hypertension and dietary athersclerosis w/o effecting cholesterol levels (was this the problem with the GLITzares ask Dina)PPAR-gamma agonists • UCP1 expression also causes INCREASED Superoxide expression in the vasculature = oxidative stress-induced vascular damage.

18. Uncoupling Protein 2 (UCP2) • UCP1 homologue, but expressed in white adipose tissues of humans during feeding, rather than brown adipose tissue of hibernating animals. Widely expressed among mammals. • UCP2 ubiquity in human tissue including macrophage-rich tissues • Critical increase (with UPC3) in metabolic adaptation to fasting regardless of weight (lean or obese) • Fetal hepatic hematopoeisis in Kupffer cells until birth due to UCP2 • Possibly critical for macrophage differentiation • Essentially Non-Thermogenic un-coupling • Still induced by superoxide and “anti-oxidant” by negative feedback • UPC homologue (with UPC3) of the skeletal muscle • Inducible neuroprotective protein by cellular redox of apoptotic suppression • Extensive expression in Spleen, Lung and macrophages suggests immunity or inflammatory dampening of ROS defenses • Negative regulation of insulin secretion in Pancreatic Beta -cell contributing significantly to Type 2 Diabetes induced by superoxide generation

19. Uncoupling Protein 3 (UCP3) • Found ONLY in skeletal muscle in humans (with UCP2) • More homologous to UCP2 than to UCP1, and un-affected by cold • And yet: possibly thermogenic in skeletal muscle based on function in yeast cells • Critical increase (with UPC2) in metabolic adaptation to fasting regardless of weight (lean or obese). Player in energy balance • Increased expression due to fatty acid-induced Superoxide generation from mitochondria. Inhibited by purine nucleotide. • Not primarily conserved in humans for uncoupling purposes (?) and not a major determinant of metabolic rate • Increased by fatty acid entry into mitochondria beyond its capacity for beta-oxidation. May be translocating fatty acids out of the matrix • Possiblle role in “proton partitioning” (reproductive vs. repair ?) • UCP3 overexpression may lead to hyperphagia without co-incident weight gain • Muscle UPC3 increased by thyroid hormone and fasting • Increased ROS generation associated with diminished UPC3 uncoupling effects. Yet still not required for bodyweight regulation, exercise tolerance, fatty-acid oxidation, or cold-induced thermogenisis • Important in MDMA-induced hyperthermia

20. PGC-1 “peroxisome proliferator-activated receptor-Gamma” Co-activator One • Nuclear protein whose expression is induced by COLD (through adrenergic agonism), hi-caloric diets, and refeeding following fasting • Induced within Brown Adipose Tissue and Skeletal muscle CAUSES: • Activates PPAR-Gamma to “proliferate peroxisomes” • UCP-1 upregulation or Adaptive Thermogenesis • Upregulatiiion for Genes of Oxidative-Phosphorylation • Stimulates DOUBLING of Mitochondrial content through NRF-1 and mtTFA PGC-1 links the external environment (cold, diet, oxygen, environmental stressors) with mitochondrial biogenesis thereby constituting the Adaptive Interface between outside and inside. (Wu Z, Puigserver P, et al. Cell 1999)

21. Peroxisomes ??

22. Reactive Oxygen Species (ROS) Reactive Oxygen Species: Radical: SuperOxide (O2-) Hydroxyl (OH-) Peroxyl (RO2) Hydroperoxyl (HO2-) NoN-Radical: Hydrogen Peroxide (H2O2) Ozone (O3) Reactive Nitrogen Species: Radical: Nitric Oxide (NO) Nitrogen Dioxide (NO2-) NoN-Radical: Peroxynitrite (ONOO-) SuperOxide  Nitric Oxide  Peroxynitrite

23. Anti-Oxidants “Any substance that, when present at low concentrations compared to those of an oxidisable substrate, significantly delays or inhibits oxidation of this substrate” Halliwell Biochem Pharmacol 1995 • Endogenous Anti-Oxidants • Glutathione • Catalase • SuperOxide Dismutase • CoEnzyme Q-10 • Cytochrome C Peroxidase • Glutathione peroxidase Dietary Anti-oxidants • Vitamins A, C, E • Alpha-Lipoic Acid • N-acetyl cysteine • Polyphenols • Green Tea (camellia sinensis) • Olive Oil (Olea europaea) • Proanthocyanidins: • Red wine (Vitis vinifera) • Blueberries (Vaccinium spp.) • Chocolate (Theobroma Cacao) • Resveratrol (Japanese Knotweed ) • Ginsengs (Panax quinguefolius, Panax ginseng)

24. Mediterranean Miracle ?…

25. Olive Oil( Olea europaea ) • Mono-Unsaturated Fat (oleic acid), + antioxidant polyphenols • THE Principle source of fat in Spain, Greece, and Italy • Olive oil can reduce the oxidative effects of fried fats on phopholipid membrane rancidity (C-C breakage) • A Natural JUICE (while all other oils from seed must be refined) Positive effects on: • Anti-oxidant Profile with Phenols, Carotenes, Vit E • Decreased Triacylglycerols • Increased HDL • Anti-Thrombotic, w/ decreased platelet -aggregations [International Consensus Report 2005]

26. 4 Groups of Rats were fed a 40% fat diet (either beef tallow, palm oil, olive oil, sunflower oil) to investigate the effects of “non-shivering thermogenesis” according to UPC mRNA expression: RESULTS: UCPs 1/2/3 were all upregulated in BAT (brown adipose tissue) in the olive oil group UCP3 in the Skeletal muscle was significantly more upregulated in the Olive oil group Saturated Fat was associated with increased food intake No significant differences in Insulin, glucose, T3, corticosterone, or Fatty acids were seen among the groups. [ Rodriguez 2002 ]

27. PROANTHOCYANIDINS In Vino Veritas

28. RED, RED WINE • In diabetics, Polyphenolics from De-alcoholized muscadine grape wine still demonstrated: •  blood glucose •  insulin •  glycated hemoglobin •  Vitamin C and E levels •  monounsaturated fatty acid cell content •  hepatic functioning (Banini,Nutrition 2006) • Red Wine Consumption (360 ml/day x 2 wks) resulted in 43% improvement in glucose disposal compared to controls (Napoli 2005) • Contains Resveratrol………..

29. Resveratrol • Now considered to be a MINOR, not “major” ingredient of Red Wine • Has recently been shown to Mimic the effects of Caloric Restriction: • Energy shunt towards tissue repair (anti-oxidation) vs. reproduction • 30% fewer calories approximates a 30% increase in LifeSpan* • 2000-2100 calories per day but untested in Humans • May be more beneficial than exercise • SIRT1 genes co-ordinate the Repair Response: turning on Sirtuins • SIRT genes 3, 4, 5 are in the mitochondria; 6 & 7 are in the nucleus • SIRT 1 genes increase the # of mitochondria produced by neurons • Sirtuins (their proteins) activate PGC-1 • PGC-1 may also generate extra chemicals to detoxify free radicals(st.pierre) • Fasting induces deacetylation of PGC-1 BY SIRT1 Fatty Acid oxidation • “It is a proven artifact that Resveratrol activates sirtuins” --Dr. DiStefano, Elixir • “Resveratrol…. : • Improves health and survival of mice on a high-calorie diet” (Baur etal, Nature 2006) • Improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1” (Lagouge, Auwerx, et.al., Cell 2006) • [as “Sirtuin Activators] mimic caloric restriction and delay aging in metazoans” (Wood JG, Rogina B, Sinclair D, et.al. Nature 2004)

30. Japanese Knotweed( Polygonum cuspidatum )

31. Knotweed as Invasive Weed (US)(polygonum cuspidatum) USDA Plant Database

32. “Food of the Gods”….

33. Chocolate !!( Theobroma cacao ) • DIABETES • Significant reduction in Blood Glucose and fructosamine levels following consumption (1% dietary) of Cacoa Liquor Proanthcyanidin (CLPr) equivalent to 2.5 Kg [Makoto 2007] • Flavonols in dark, not WHITE!, chocolate of daily single bar 100 g (500 mg polyphenols) for 15 days increased insulin sensitivity (with co-incident decrease in blood pressure) measured by HOMA-IR [Grassi 2005 Am J Clin Nutr.] • Dark Chocolate flavonols decreased Insulin resistance, LDL cholesterol ; improved flow-mediated dilation ; and ameliorated insulin sensitivity in patients with essential hypertension [Grassi 2005 Hypertension ] Cacoa or dark-chocolate has been shown to reduce the cardiovascular risks of • Atherosclerosis • Hypertension • Platelet adhesion • LDL Oxidation

34. Apple Pie Ingredient…

35. CinnamonCinnamomum zeylanicum • Cinnamon ( C. cassia ) at daily intake of 1, 3, or 6 grams for 40 day duration led to decrease serum glucose levels of 18-29% • Not dose Dependent (all doses were equipotent) • Also, Decreased serum triglyceride levels of 23-30% • Phenolic acids in C. zeylanicum provide even higher TEAC* concentrations [Khan et.al. 2003] * Total Equivalent Anti-oxidant Concentrations “A compound in cinnamon called methylhydroxy chalcone polymer (MHCP) makes fat cells more responsive to insulin by activating an enzyme that causes insulin to bind to cells and inhibiting the enzyme that blocks this process”. -- Dr. Richard A Anderson USDA [Jarvill-Taylor KJ, et.al. 2001]

36. Panax Quinquefolius

37. American Ginseng*( Panax quinquefolius ) • Anti-oxidant Ginsenosides identical to research conducted on the Korean Cousin (Panax ginseng) • Over 2000 published articles • Ancient Chinese indication for “quenching thirst” • Rank order of ginsenoside content: leaf > berry > root (sustainable) • Numerous studies demonstrating anti-hyperglycemic effects and improved insulin resistance • Suspected pancreatic ß-cell sparing effect ( Xie, 2005) “KING ROOT” ( avg $ / Pound = $400 ) * Endangered - Habitat destruction, , Housing development, Mountain Top Removal

38. Symbiosis Mitochondrion Chloroplast CO2

39. Mitochondria in the Modern Mythos Midi-chlorians ( Mitochondria & Chloroplasts ) The boy nodded his understanding. "Can I ask you something?” The Jedi Master nodded. "What are midi-chlorians?" Wind whipped at Qui-Gon's long hair, blowing strands of it across his strong face. "Midi-chlorians are microscopic life-forms that reside within the cells of all living things and communicate with the Force.” “They live inside of me?" the boy asked. "In your cells.…. We are symbionts with the midi-chlorians." "Symbi-what?" "Symbionts. Life-forms living together for mutual advantage. Without the midi-chlorians, life could not exist, and we would have no knowledge of the Force. Our midi-chlorians continually speak to us, Annie, telling us the will of the Force." "They do?" Qui-Gon cocked one eyebrow. "When you learn to quiet your mind, you will hear them speaking to you." Anakin thought about it for a moment, then frowned. "I don't understand." Qui-Gon smiled, and his eyes were warm and secretive. "With time and training, Annie, you will." ---from “Star Wars Episode I: The Phantom Menace” novelization by Terry Brooks

40. THE END May the Force Be With You

42. Remember the waterwheel

43. Blueberries( Vaccinium myrtillus )

44. Adrenalin stress types, under sympathetic nervous system stress, will trigger peripeheral coversions of Thyroxine to metabolically active T3 resulting not only in an upregulation of a basal metabolic rate but the increased expression of of all three uncoupling proteins (Ricquier Biochem Journal on UCPs) ( Masaki FEBS letter 1997)( Gong DW T3 B3 adrenergic agonists J Biol Chem 1997) SAVE THIS!PPAR -gamma can be upregulated by oleic acid perhaps:this could likely lead to upregulation of UCPs in the BAT (as thiazollidinediones upregulate UCP3 in adipose tissue yet unfortunately down regulate UCP2 in skeletal muscle)